Method and apparatus for fractional separation of proteins from plant material

ABSTRACT

A method of extracting and purifying recombinant protein(s) from transgenic plant matter is disclosed. Fractioning of juice that has been extracted from the plant matter is obtained by using a multiple stage filtering process that uses multiple stages of decreasing porosity (preferably screening) followed by preferably membrane type filters, ion exchange, membrane adsorber, and chromatographic processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part of co-pending U.S. patent application Ser. No. ______, filed Apr. 10, 2001, entitled “Sugarcane Fractioning System”, which is incorporated herein by reference.

[0002] Priority of U.S. Provisional Patent Application Serial No. 60/196,085, filed Apr. 11, 2000, which is incorporated herein by reference, is hereby claimed.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This work was supported by DOD Grant No. DAAG55-97-1-0096. The government may have rights in this invention.

REFERENCE TO A “MICROFICHE APPENDIX”

[0004] Not applicable

BACKGROUND OF THE INVENTION

[0005] 1. Field of the Invention

[0006] This invention relates to a novel technology for the separation of proteins from transgenic plant matter (eg. stalk, leaf and/or grain) and fractional purification of proteins so that proteins can be separated and further purified from other compounds. The invention allows for the rapid separation and fractional purification of large quantities of proteins. The present invention more particularly relates to a separation and fractional purification process that can be applied with any type of transgenic vegetal plant material (including, eg. plant stalk, leafy material and/or grain) such as but not limited to cane, barley, corn, potatoes, alfalfa, used to produce essentially any category of recombinant protein(s) such as but not limited to monoclonal antibodies (MAB), lectins, collagens, enzymes, or therapeutic proteins. During each step or any of the steps of this novel process, unconventional or conventional laboratory analysis could be performed in order to monitor the streams and the concentration of the protein(s) of interest.

[0007] 2. General Background of the Invention

[0008] The extraction of the protein(s) of interest from the feedstock can be performed through different means that will not be destructive to the different proteins. The extraction can be performed through a preparation or comminuting step for size reduction of the feedstock followed by maceration or leaching steps. Comminuting of the feedstock will be done with apparatus such as but not limited to crusher, grinder, and cutting machine. For example, starting with genetically modified sugarcane stalks containing the protein(s) of interest, and passing the stalks through a pressure system such as roller/crusher allows extracting a liquid in this case also called pression juice, which contains the protein(s) of interest.

[0009] The present invention relates to processing of plant matter for the recovery (fractional purification) of high value proteins so that these proteins can be separated and further purified from other compounds.

[0010] The present invention enables the rapid separation and fractional purification of large quantities of proteins. This process is preferably applied to any type of transgenic vegetable plant material such as, for example; cane, sugarcane, barley, corn, potatoes, alfalfa, etc., and can be used to produce essentially any category of recombinant protein(s) such as, but not limited to, monoclonal antibodies (MAB), lectins, collagens, enzymes, or therapeutic proteins. During each step or any of the steps of the process of the present invention, unconventional or conventional laboratory analysis could be performed in order to monitor the streams and the concentration of the protein(s) of interest.

BRIEF SUMMARY OF THE INVENTION

[0011] The present invention provides a process for extracting high value protein(s) directly from transgenic plant material.

[0012] The extraction of transgenic plant feedstock containing the protein(s) of interest can be performed through different means that will not be destructive to the different proteins. Genetically modified and/or non-genetically modified plants containing the protein(s) of interest and able to produce a juice when passing through a pressure system can be processed. For example, starting with genetically modified plant material containing the protein(s) of interest, and passing the plant matter through a pressure system such as roller/crusher allows extracting a liquid, in this case also called “pression juice”, which contains the protein(s) of interest.

[0013] The extracting pressure system can for example use: (a) different geometry rollers, (b) a plurality of rollers, (c) water for further extraction of pression juice, (d) series pressure system where the plant matter after the first pressure system will feed a second pressure system in series with the first pressure system, etc. (e) a buffer solution which will avoid partially or entirely, oxidation or degradation of some compounds contained in the pression juice.

[0014] In order to improve the juice extraction of the pressure system, the plant matter can be previously shredded. Both shredder and pressure system are preferably part of the extraction step. The extraction step can work continuously or discontinuously. Extraction could also be performed through a leaching process such as diffusion.

[0015] Following the extraction step, the pression juice is transmitted, either by gravity or by means of pumping to a screening system that is preferably composed of one or several screening steps. For example, a three step screening system can be used that is comprised of: (a) a first screening step that removes selected particulate, eg. matter larger than about 500 microns to 1000 microns, (b) the second screening step can be used for removing particulate size larger than about 150 microns to 250 microns, (c) the third screening step can be used to remove particulate size larger than about 10 to 60 microns.

[0016] The screening system can include, for example, screens that are stationary, vibrating, rotary or any combination of these types of screens. Screens could also be self-cleaning units. The screened juice is recovered for further processing and the reject is discarded or sent to alternate processing. Press filter(s) or other filtering devices such as pressure filters could be used as an option to the screening step.

[0017] The screened juice is transferred to a receiving/mixing tank where its pH is adjusted to a value that is preferably in the range of between about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank could be equipped with a low shear rate-mixing device. The tank is preferably designed to control the temperature of the juice to a value between about 4° Celsius to 70° Celsius.

[0018] The juice from the receiving/mixing tank is transmitted (eg. pumped) at constant flow into a first membrane separation system. This first system performs the separation of suspended solids with a size larger than between about 0.1 to 0.2 microns. The clean juice contains the protein(s) of interest. This clean juice or “first permeate fraction” is sent to a receiving tank before transmission to the next method step.

[0019] The membrane reject or first retentate fraction is discarded or sent to alternate processing. The first retentate fraction contains contaminants such, as but not limited to: dextrans, waxes, bagacillo, bacterias, yeast, and suspended solids that are typically larger than about 0.1 to 0.2 microns. Membranes can be of different types, materials and configurations. As an example, hollow fiber polymeric membranes can be used. However, composite membranes can be used as well as inorganic (for example, ceramic and coated stainless steel tube membranes) and polymeric membranes with different, selected configurations.

[0020] The first membrane separation system can be comprised of a single or several membranes working in parallel or in series. Operating temperature is preferably in the range of between about 4° Celsius to 70° Celsius. Fluxes obtained are preferably in the range of between about 15 to 160 gfd (gallon per square foot per day) at different trans-membrane pressures. During this step some properties of the membrane such as hydrophilicity can enhance the separation process.

[0021] The permeate (also called clean fraction from the first step membrane) is collected into a tank called first permeate tank.

[0022] The product from the first fraction tank is used to feed at preferably constant flow, the second membrane separation system. This second membrane separation system performs the separation of particulate larger than between about 0.01 to 0.05 microns. The permeate fraction is collected into a tank called the second fraction tank. The retentate fraction is collected into a tank called second retentate tank. According to its (their) molecular size(s), the protein(s) of interest could be in either the second retentate fraction or the second permeate fraction.

[0023] Membranes can be of different types, materials and configurations. Hollow fiber polymeric membranes can be used. However, composite membranes can be used as well as inorganic (ceramic and coated stainless steel tube membranes) and polymeric membranes all of them with arrangement including hollow fiber, spiral, plate and tubular module configurations.

[0024] The second membrane separation system can be composed of a single or several membranes working in parallel or in series. Operating temperature is preferably in the range of between about 4° Celsius to 70° Celsius. Fluxes can be in the range of between about 5 to 80 gfd (gallon per square foot per day) at different transmembrane pressures. The system can be hydraulically designed in order not to exceed a shear rate of 10,000 sec⁻¹. During this step some properties of the membrane such as hydrophilicity can enhance the separation process. The discarded fraction is sent to alternate processing.

[0025] The fraction containing the protein(s) of interest, either the second permeate fraction or the second retentate fraction is collected into a second fraction tank. From the second fraction tank, the second fraction is transmitted (eg. pumped) at preferably constant flow into the third membrane separation system, which has cut size of about 5,000 to 80,000 molecular weight.

[0026] The membranes used in the third separation system can be made of different material with different shape and configuration. Membranes can be of different types, materials and configurations. The membrane used can be a flat plate configuration, often referred to as “cassettes”. However, hollow fiber and spiral wound membranes could also be used. Different materials either regenerated cellulose or polyethersulfone membranes can be used. Other materials that could be used such as polymeric membranes with arrangement including hollow fiber, spiral, plate or tubular module configurations.

[0027] The third membrane separation system can be comprised of a single or several membranes working in parallel or in series. Operating temperature is preferably in the range of between about 4° Celsius to 70° Celsius. Fluxes can be in the range of about 0.1 to 30 gfd (gallon per square foot per day) at different transmembrane pressures. The system can be hydraulically designed in order not to exceed a shear rate 10,000 sec⁻¹.

[0028] The third membrane separation system produces two fractions: (a) the third permeate fraction and (b) the third retentate fraction. The protein(s) of interest is (are) in one of these two fractions. The discarded fraction is sent to alternate processing.

[0029] The fraction containing the protein(s) of interest is collected into a third fraction tank prior to any further treatment step during the purification process. The third fraction tank is a receiving/mixing tank where the pH of the fraction is adjusted to a value in the range of between about 5.2 to 8.3, accordingly to the protein(s) of interest. The third fraction tank can be equipped with a low shear rate-mixing device. The third fraction tank can also be temperature controlled to maintain the temperature of the juice to a value between about 4° Celsius to 70° Celsius.

[0030] The protein fraction of interest after pH adjustment is transferred (eg pumped) at a rate of about 0.5 to 3.0 beds volume per hour through an ion exchange column containing a weak anionic resin with higher affinity (at this pH of about 4.5 tp 8.3, preferably 5.2 to 8.3) for colorants than any other compounds. Temperature during this step is maintained at a value between about 4° Celsius to 70° Celsius. Decoloration of the incoming feed is between about 25% and 95%.

[0031] The decolorized fraction containing the protein(s) of interest is collected into a ion product tank where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The ion product tank could be equipped with a low shear rate-mixing device. The ion product tank could also be designed to control the temperature of the juice to a value between about 4° Celsius to 70° Celsius. The juice from this ion product tank is transferred (eg. pumped) at a rate of about 0.1 to 3.0 beds volume per hour through an ion exchange chromatographic process for further purification. The ion exchange chromatographic process step produces several fractions, one of them with higher concentration of the protein(s) of interest. A membrane adsorber could replace the ion exchange chromatographic step.

[0032] The resulting fraction containing the protein(s) of interest is collected into an ion exchange chromatographic receiving/mixing tank where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The ion exchange chromatographic tank could be equipped with a low shear rate-mixing device. The ion exchange chromatographic tank could also be temperature controlled to maintain the temperature of the juice to a value between about 4° Celsius to 70° Celsius. The fraction of the protein(s) of interest could be sent to a concentration step such as a low temperature evaporating system for further concentration such as flash/freeze dry. The product from the concentration step (eg. evaporation station) contains the fractionated desired protein(s) partially purified and concentrated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] For a further understanding of the nature, objects, and advantages of the present invention, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein:

[0034]FIG. 1 is a schematic flow diagram illustrating the preferred embodiment of the method and apparatus of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0035]FIG. 1 is a schematic diagram of the preferred embodiment of the apparatus of the present invention, designated generally by the numeral 10. FIG. 1 also shows the various method or process steps of the preferred embodiment of the apparatus of the present invention. Transgenic plant processing system 10 receives plant feedstock 11 that are treated preliminarily by extraction step 13. The feedstock 11 can be any type of transgenic plant material such as eg. cane, sugarcane, barley, corn, potatoes, alfalfa. As used herein, the terms “transgenic material should be construed to include any plant material such as leafy material, stalks, grain, or the like.

[0036] The extraction step 13 can start with genetically modified plant matter 11 containing the protein(s) of interest, and passing the stalks through a pressure system such as roller/crusher allows extracting a liquid in this case also called pression juice 14, which contains the protein(s) of interest.

[0037] The extracting pressure system or extraction step 13 can use: (a) different geometry rollers, (b) any quantity of rollers, (c) water 12 for further extraction of pression juice 14, (d) series pressure system where the plant matter 11 after the first pressure system will feed a second pressure system in series with the first pressure system, etc. (e) a water and/or buffer solution 12 which will avoid partially or entirely oxidation or degradation of some compounds contained in the pression juice 14.

[0038] In order to improve the juice extraction of the pressure system, the plant matter 11 can be previously shredded. Both shredder and pressure system are part of the extraction step 13. The extraction step 13 can work continuously or discontinuously.

[0039] Extraction 13 could be also performed through a leaching process such as diffusion.

[0040] Following the extraction step 13, the pression juice 16 feeds, either by gravity or by means of pumping to a screening system composed of one or several screening steps 17, 19, 21. For example, a three steps screening system will be composed of: (a) the first screening step 17 could remove particulate matter larger than about 500 microns to 1000 microns, (b) the second screening step 19 used for particulate size larger than about 150 microns to 250 microns, (c) the third screening step 21 removing particulate size larger than about 10 to 60 microns.

[0041] Screens 17, 19, 21 could be stationary, vibrating, rotary or any combination of these types of screens. Screens 17, 19, 21 could also be self-cleaning units. The screened juice is recovered at mixing tank 24 for further processing and the reject 18, 20, 22 is discarded or sent to alternate processing.

[0042] Press filter(s) or other filtering devices such as pressure filters could be used as an option to the screening step comprised of screens 17, 19, 21.

[0043] The screened juice is transmitted to receiving/mixing tank 24 where its pH is adjusted to a value preferably in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank 24 can be equipped with a low shear rate-mixing device. The tank 24 can also be temperature controlled to maintain a temperature of the juice to a value about 4° Celsius to 70° Celsius.

[0044] The juice from the receiving/mixing tank 24 is transmitted (eg. pumped) at constant flow into a first membrane separation system 25. This first membrane separation system 25 performs the separation of suspended solids with a size larger than about 0.1 to 0.2 microns. The clean juice contains the protein(s) of interest. This clean juice or first permeate fraction is sent to first fraction tank 27 before going into the next step. The membrane reject or first retentate fraction 26 is discarded or sent to alternate processing.

[0045] The first retentate fraction 26 contains contaminants such as but not limited to: dextrans, waxes, bagacillo, bacterias, yeast, and suspended solids larger than 0.2 microns. Membranes that are used in system 25 can be of different types, materials and configurations. Hollow fiber polymeric membranes can be used; however, composite membranes can be used as well as inorganic (ceramic and coated stainless steel tube membranes) and polymeric membranes all of them with different configurations. The first membrane separation system 25 can be comprised of a single or several membranes working in parallel or in series. Operating temperature is preferably in the range of about 4° Celsius to 70° Celsius. Fluxes can be in the range of about 15 to 160 gfd (gallon per square foot per day) at different trans-membrane pressure. During this step at first membrane separation system 25 some properties of the membrane such as hydrophilicity can enhance the separation process. As previously indicated, the permeate also called clean fraction from the first step membrane is collected into a tank 27 called first fraction tank.

[0046] The product from the first fraction tank 27 is used to feed (at preferably constant flow) the second membrane separation system 28. This system 28 performs the separation of particulate larger than about 0.01 to 0.05 microns. The permeate fraction 29 is collected into a tank called second permeate fraction.

[0047] The retentate fraction 30 is collected into a tank called second retentate tank. Accordingly to its (their) molecular size(s), the protein(s) of interest can be either into the second retentate fraction 30 or the second permeate fraction 29. Membranes in the second membrane system 28 can be of different types, materials and configurations. Hollow fiber polymeric membranes can be used: However, composite membranes can be used as well as inorganic (ceramic and coated stainless steel tube membranes) and polymeric membranes all of them with arrangement including hollow fiber, spiral, plate and tubular module configurations. The second membrane separation system 28 can be composed of a single or several membranes working in parallel or in series.

[0048] Operating temperature is preferably in the range of a value about 4° Celsius to 70° Celsius. Fluxes can be in the range of about 5 to 80 gfd (gallon per square foot per day) at different transmembrane pressure. The second membrane separation system 28 is hydraulically designed in order not to exceed a shear rate of 10,000 sec⁻¹. During this step some properties of the membranes 28 such as hydrophilicity can enhance the separation process. Any discarded fraction can be sent to alternate processing.

[0049] The fraction containing the protein(s) of interest can be either the second permeate fraction 29 or the second retentate fraction 30 and is collected into the second fraction tank 31. From the second fraction tank 31, the second fraction is transmitted (eg. pumped) at preferably constant flow into the third membrane separation system 32, which has cut size of about 5,000 to 80,000 molecular weight.

[0050] The membrane(s) used in the third separation system 32 can be made of different material with different shape and configuration. Such membranes can be of different types, materials and configurations. The membrane(s) can be flat plate configuration, often referred as cassettes. However, hollow fiber and spiral wound membranes can be used. Different materials such as either regenerated cellulose or polyethersulfone membranes can be used. Other materials can be used such as eg. polymeric membranes with arrangement including hollow fiber, spiral, plate or tubular module configurations.

[0051] The third membrane separation system 32 can be comprised of a single or several membranes working in parallel or in series. Operating temperature is in the range of a value about 4° Celsius to 70° Celsius. Fluxes can be in the range of about 0.1 to 30 gfd (gallon per square foot per day) at different transmembrane pressure. The third membrane separation system 32 can be hydraulically designed in order no to exceed a shear rate 10,000 sec⁻¹. The third membrane separation system 32 produces two fractions: (a) the third permeate fraction 33 and (b) the third retentate fraction 34. The protein(s) of interest is (are) in one of these two fractions 33, 34. Any discarded fraction can be sent to alternate processing.

[0052] The fraction containing the protein(s) of interest is collected into the third fraction tank 35 prior to any further treatment step during the purification process. The third fraction tank 35 is preferably a receiving/mixing tank where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank 35 can be equipped with a low shear rate-mixing device. The tank 35 can also be temperature controlled to maintain the temperature of the processed juice in the tank 35 to a value about 4° Celsius to 70° Celsius. The protein fraction of interest after pH adjustment is transferred (eg. pumped) at a rate of about 0.5 to 3.0 beds volume per hour through an ion exchange column 36 containing a weak anionic resin with higher affinity at this pH of about 5.2 to 8.3 for colorants than any other compounds. Temperature during this step at column 36 is maintained at a value about 4° Celsius to 70° Celsius. Decoloration of the incoming feed is between about 25% and 95%.

[0053] The decolorized fraction containing the protein(s) of interest is collected into an ion product receiving/mixing tank 38 where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank 38 can be equipped with a low shear rate-mixing device. The tank 38 can also be temperature controlled to maintain a temperature of the juice to a value about 4° Celsius to 70° Celsius. The juice from tank 38 is transferred (eg. pumped) at a rate of about 0.1 to 3.0 beds per volume through an ion exchange chromatographic process step 39 for further purification. The ion exchange chromatographic process step 39 produces several fractions, one of them with higher concentration of the protein(s) of interest. Membrane adsorber could replace the ion exchange chromatographic step 39.

[0054] The resulting fraction containing the protein(s) of interest is collected into an ion exchange chromatographic receiving/mixing tank 41 where the pH of the fraction is adjusted to a value in the range of about 5.2 to 8.3, accordingly to the protein(s) of interest. The tank 41 can be equipped with a low shear rate-mixing device. The tank 41 can also be temperature controlled to maintain a temperature of the juice to a value about 4° Celsius to 70° Celsius. The fraction of the protein(s) of interest could be sent to a low temperature concentration step 42 (eg. evaporating system) for further concentration. Such a concentration step can be, for example, a flash/freeze dry step. The product from the concentration step 42 (evaporation station) contains the fractionated protein(s) 44 partially purified and concentrated.

[0055] The following is a list of suitable parts and materials for the various elements of the preferred embodiment of the present invention. PARTS LIST PART NO. DESCRIPTION 10 plant matter fractioning system 11 plant matter feedstock 12 water/buffer 13 extraction step 14 pression juice 15 reject (bagasse) 16 flowline 17 first screen 18 reject 19 second screen 20 reject 21 third screen 22 reject 23 ph buffer 24 mixing tank 25 first membrane 26 first retentate fraction 27 first fraction tank 28 second membrane 29 second permeate tank 30 second retentate fraction 31 second fraction tank 32 third membrane 33 third permeate 34 third retentate 35 third fraction tank 36 ion exchange 37 rejects 38 ion product tank 39 ion exchange chrom 40 reject 41 ion exchange chrom tank 42 concentration step 43 condensates 44 partially purified protein

[0056] The foregoing embodiments are presented by way of example only; the scope of the present invention is to be limited only by the following claims. 

1. A method of refining transgenic plant matter to extract protein matter comprising the steps of: a) providing a transgenic plant matter feedstock to be processed; b) extracting the plant matter feedstock to extract pression juice; c) cleaning the pression juice to remove particulate matter; d) adjusting the pH of the juice to a pH of at least 4.5; e) transmitting the juice from steps “a” through “d” to a membrane separation system in order to produce two fractions, one of the fractions containing a protein of interest:
 2. The method of claim 1 further comprising the step of removing colorant from the juice with ion exchange.
 3. The method of claim 1 wherein there is no fermentation involved for the product or separation of protein.
 4. The method of claim 1 wherein the screen system has a porosity of between about 60-500 microns.
 5. The method of claim 1 wherein the juice pH value is adjusted to a range of between about 5.2 and 8.3.
 6. The method of claim 1 wherein in step “c” there are at least one filter or multiple screens that include multiple screens having different porosities.
 7. The method of claim 1 wherein the membrane separation system of step “e” wherein there are a plurality of membranes.
 8. The method of claim 1 wherein there are a plurality of membrane separation stations in step “e” and a plurality of fraction tanks, one fraction tank for each membrane separation station.
 9. The method of claim 7 wherein there are at least three membrane separation stations.
 10. The method of claim 7 wherein each membrane separation station generates a retentate fraction and a permeate fraction.
 11. The method of claim 12 wherein there are a plurality of fraction tanks and each permeate fraction is transmitted to a tank.
 12. The method of claim 1 wherein the transgenic plant matter is barley.
 13. The method of claim 1 wherein the transgenic plant matter is corn.
 14. The method of claim 1 wherein the transgenic plant matter is potatoes.
 15. The method of claim 1 wherein the transgenic plant matter is alfalfa.
 16. A process for extraction of protein from transgenic plant matter, comprising the steps of: a) extracting juice from a feedstock of protein contained in the transgenic plant matter; b) preliminarily screening said juice to remove particulate matter, using a plurality of screens spanning a range of porosity of between 50 and 500 microns porosity; c) treating the juice with multiple stages of ultra-clarifying filtration of decreasing porosity, including some ultra-clarifying filtration that includes membrane separation.
 17. The process of claim 16 wherein the step of ultra-clarifying the juice comprises ultra-filtering the juice with a membrane having a cutoff that will fractionate proteins having molecular weight between about 5,000 and 500,000.
 18. The process of claim 16 wherein the transgenic plant matter is barley.
 19. The process of claim 16 wherein the transgenic plant matter is corn.
 20. The process of claim 16 wherein the transgenic plant matter is potatoes.
 21. The process of claim 16 wherein the transgenic plant matter is alfalfa. 